1. Field of the Invention
This invention is related to the field of veterinary diagnostics and, more particularly, to a homogeneous fluorescence polarization-based assay to detect specific antibodies contained in the blood of horses and ponies infected with the lentivirus, aetiologic for Equine Infectious Anemia (EIA).
2. Description of Related Art
Equine Infectious Anemia Virus (EIAV) is a lentivirus genetically related to human immunodeficiency virus type 1 (HIV-1) that infects horses, ponies, and other equids (for a recent review see Montelaro, et al., xe2x80x9cEquine Retroviruses, in J. A. Levy, Ed., The Retroviridae, Vol. 2, p. 257 (Plenum Press: 1993 N.Y.). It causes a chronic disease characterized by a period of cyclic fevers and viremia, followed by clinical quiescence. The animals generally survive this disease but remain infected, becoming lifelong inapparent carriers; they appear to be healthy but in fact still may have virus in their blood There are thousands of EIAV-positive horses in the US; most of them reside in the xe2x80x9chot zonexe2x80x9d, a group of 18 states along the Gulf coast and Mississippi valley (see Cordes, xe2x80x9cEquine Infectious Anemiaxe2x80x9d, USDA 91-55-032 (1996)). The disease is most prevalent there due to the humid environment that favors growth of horse flies, the major vector of transmission of EIAV. In an attempt to control the spread of this virus, horses are tested before showing, breeding, or crossing state lines. If a horse is found to be seropositive, its movement is severely restricted; the horse must be euthanized or quarantined with a 200-yard barrier for the rest of its life. However, because testing is not yet mandatory for all horses, it is estimated that over 80% have never been tested; this pool of horses may be a major reservoir for the virus. Efforts are underway to encourage, and in some states mandate, testing of all equids to better control this disease and reduce the rate of infection.
EIAV-infected animals mount a vigorous immune response to the viral infection. This results in reduction of viremia during clinical quiescence to very low, often undetectable, levels. This immune response is characterized by high-titer antibodies directed to three major viral antigens: the envelope glycoproteins, gp90 and gp45, and the capsid protein or core antigen, p26. Due to the presence of high levels of antibody and low levels of virus during most of the disease course, diagnostic assays have focused on detection of viral antibodies.
One way to improve testing compliance is to develop better, faster assays. Current official diagnostic assays for EIAV include agar gel immunodiffusion (AGID) as reported in Coggins, et al., Cornell Vet USA LX: 330 (1970), competitive ELISA (C-ELISA), and synthetic antigen ELISA (SA-ELISA). The first two assays detect antibodies to the major core protein p26, which has a well conserved structure but is a relatively poor immunogen compared to the envelope proteins, gp90 and gp45. SA-ELISA detects antibodies to gp45 and is approved for use, but can have a lower sensitivity. The major drawbacks of the AGID test are the length of time it takes to test the samples and the technical difficulty in interpreting the results. ELISA-based tests can be completed in several hours, but in a recent study the C-ELISA had a 2% false positive rate, as reported in Issel, EIA-Hotzone Project, U of Kentucky.
Fluorescence polarization (FP) has been used as a tool to monitor protein-protein, protein-peptide; and other intermolecular interactions, as described in Jolley, J. Biomol, Screen 1: 33 (1996). First described by Perrin (1926), it is the property of many fluorophores that they emit light in the same direction in which it is absorbed. When a fluorophore is freely rotating in solution, the light is emitted in all directions by virtue of the molecule""s rotation during the lifetime of the fluorescence emission; it is non-polarized. If, however, the fluorophore is part of a slowly rotating molecule (one that is large or in a viscous environment), the molecule does not rotate quickly with respect to the lifetime of the fluorescence, and the emission will occur in roughly the same direction as the absorption; it is polarized. This property of fluorescence can therefore be used to distinguish small molecules (e.g. fluorescent-labeled peptides) from large ones (e.g. peptide bound to antibody). Relatively recent advances in instrumentation have allowed the use of this phenomenon to develop rapid immunoassays; for a large number of analytes including therapeutic drugs and metabolites as well as antibodies to infectious agents as, for example, Nielsen, et al., J. Immunol. Methods 195: 161 (1996). These assays can be performed in a matter of minutes (vs. hours or days for the other tests) and usually do not require extensive sample preparation. In addition, the materials required for the assay are relatively simple and highly stable, making this technique attractive for field use.
In light of the need for a more rapid assay that can be used in the field to detect EIAV-infected horses, we pursued FP as a medium on which to develop a new diagnostic for anti-EIAV antibodies. We selected, labeled, and evaluated several candidate peptides for their ability to detect the presence of antibodies to three EIAV proteins. This investigation has led to the development of an FP-based assay which uses a well-conserved, immunodominant region of gp45 transmembrane protein. The test is rapid and possesses both high sensitivity and very high specificity. It reacts with antibodies in serum or plasma from both experimentally- and field-infected animals from various geographic areas.
In a first principal aspect, the present invention provides a synthetic antigen probe comprising a fluorophore conjugated to a peptide comprising a sequence of amino acids selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, wherein the synthetic fluorescent antigen probe binds to serum antibodies to equine infectious anemia virus to produce a detectable change in fluorescence polarization.
In a second principal aspect, the present invention provides an assay for serum antibodies reactive to an antigen common to a number of field strains of equine infectious anemia virus that comprises the following steps. First, a serum specimen suspected of containing antibodies reactive with an antigen of equine infectious anemia virus is diluted with a buffer solution to provide a buffered specimen. Next, a synthetic fluorescent antigen probe is added to the buffered specimen. The synthetic fluorescent antigen probe comprises a fluorophore conjugated to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. The buffered specimen with added antigen probe is incubated for a time sufficient to permit binding in solution of the EIAV antibodies with the antigen probe to provide a reaction product. The fluorescence polarization of the reaction product is then compared to a blank control.
In a third principal aspect, the present invention provides a diagnostic assay kit for detecting serum antibodies reactive to a number of field strains of equine infectious anemia virus. The kit is comprised of a synthetic fluorescent antigen probe in amount suitable for at least one assay and suitable packaging. The synthetic fluorescent antigen probe comprises a fluorophore conjugated to a peptide comprising a sequence of amino acid selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
In accordance with preferred embodiments of the present invention, the fluorescence polarization-based diagnostic assay, utilizing a synthetic fluorescent antigen probe, is rapid, easy to use, and has a high sensitivity to and specificity for a number of field strains of equine infectious anemia virus.